Fig. 5: Pin1 isomerizes Ubc9 to facilitate formation of the Ubc9-SUMO1 thioester. | Nature Communications

Fig. 5: Pin1 isomerizes Ubc9 to facilitate formation of the Ubc9-SUMO1 thioester.

From: Stabilization of Pin1 by USP34 promotes Ubc9 isomerization and protein sumoylation in glioma stem cells

Fig. 5

a A diagram of the Ubc9 protein sequence depicting the potential phosphorylated serine-proline (pS-Pro) motif composed of the serine 71 and proline 72 residues. b Co-immunoprecipitation to determine the interaction between Pin1 and Ubc9 in T387 GSCs. c Cis-trans isomerization assay to determine the Pin1-catalyzed isomerization of the phosphorylated Ubc9. The phosphorylated Ubc9 oligopeptides (Ubc9-pS71) or the control peptides were incubated with the purified flag-tagged Pin1. The 4-nitroaniline released from the Ubc9 peptides was measured continually, and its change reflected the difference in the isomerization of prolyl bonds. Data represent three independent experiments (***P < 0.001; means ± s.d. two-way ANOVA). d, e Immunoblot analyses of the Ubc9-SUMO1 thioester (Ubc9 ~ SUMO1) formation in 293 T cells expressing ectopic Ubc9 and SUMO1 (d) and in T387 GSCs (e). The immunoprecipitated Ubc9-SUMO1 conjugate located at ~37 kDa. Addition of DTT (200 mM) and β-ME (5%) into the loading buffer broke the Ubc9-SUMO1 thioester, leading to the disappearance of the Ubc9-SUMO1 conjugate and the increase of free SUMO proteins at ~15 kDa. For a fair comparison for input, DTT and β-ME were added before loading to disrupt any potential thioesters. f Immunoblot analysis of the Ubc9-SUMO1 thioester (Ubc9 ~ SUMO1) formation in 293 T cells after disruption of endogenous Pin1. The immunoprecipitated Ubc9-SUMO1 conjugate was detected by anti-SUMO1 antibodies. g Immunoblot analysis of the Ubc9-SUMO1 thioester (Ubc9 ~ SUMO1) and Ubc9-SUMO2/3 thioester (Ubc9 ~ SUMO2/3) formation in T387 GSCs after disruption of Pin1. h Immunoblot analysis of the Ubc9-SUMO1 thioester (Ubc9 ~ SUMO1) formation in 293 T cells overexpressing ectopic Pin1. i Immunoblot analysis of the Ubc9-SUMO1 thioester (Ubc9 ~ SUMO1) formation in 293 T cells expressing ectopic wild type Ubc9 or the Ubc9-S71A mutant Ubc9 and SUMO1. j Immunoblot analysis of global protein sumoylation status in T387 GSCs expressing ectopic Ubc9. GSCs were transduced with the wild type Ubc9 or the Ubc9-S71A mutant through lentiviral infection followed by infection with lentiviruses carrying an shRNA targeting 3’-UTR of Ubc9 (shUbc9-3’UTR). The blotting experiments were repeated at least three times with biological replicates (b, d–j). Source data are provided as a Source Data file.

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